Thin layer chromatography (hereinafter also referred to as “TLC”) is known as a method for separating and detecting specific components in a mixture. When separating components by means of TLC, detection is achieved by subjecting spots, which are obtained by developing a sample, to irradiation with ultraviolet rays or to a coloration treatment using a coloring reagent on the basis of differences in the optical responsiveness of, for example, a separating agent layer and the components being detected.
Meanwhile, separating agents that contain polysaccharide derivatives such as phenyl esters of polysaccharides are known as separating agents for optical isomers. When used in separating agent layers for TLC plates, such separating agents that contain aromatic rings are, in some cases, unable to detect components to be detected when irradiating with ultraviolet rays or carrying out a coloration treatment using a coloring reagent.
As means for solving this type of problem, a TLC plate is known in which a first separating agent layer, which can achieve the separation of target substances but does not exhibit an optical responsiveness, and a second separating agent layer, which does not achieve a separation function but exhibits an optical responsiveness, are formed side-by-side on the same substrate (for example, see patent document 1). In this TLC plate, target substances are developed from the first separating agent layer to the second separating agent layer, spots separated by the first separating agent layer migrate to the adjacent second separating agent layer and are detected there according to their optical responsiveness.
In this TLC plate, the extract component that is readily adsorbed by the first separating agent layer does not, in some cases, satisfactorily reach the second separating agent layer. In addition, because the speed of migration of a spot of a target substance in a sample generally varies in each separating agent layer, the positional relationship of spots in a first separating agent layer is not necessarily precisely maintained in a second separating agent layer. Therefore, it is not necessarily possible to precisely detect the state of separation in the first separating agent layer in the aforementioned TLC plate, and further research is needed in this respect at least.
Patent Document 1: Japanese Patent No. 3140138
The present invention provides a chromatographic medium which can separate and detect target substances in a sample using a single kit.
The inventors of the present invention found that the aforementioned problems could be solved by using a chromatographic medium having a separating agent layer, which is used to separate target substances, and a permeation layer, which is laminated so as to face the separating agent layer and which is used to enable permeation of the target substances separated by the separating agent layer, wherein a region in which the permeation layer is not laminated, that is, a region where only the separating agent layer is present, is present on a part of the separating agent layer, a layer that exhibits a separating property for the target substances and exhibits an optical responsiveness to ultraviolet rays is used as the separating agent layer, and a layer that exhibits optical response properties that are different from those of the separating agent layer is used as the permeation layer, and thereby completed the present invention.
That is, the present invention provides a chromatographic medium having a separating agent layer, which is used to separate target substances, and a permeation layer, which is laminated so as to face the separating agent layer and which is used to enable the permeation of the target substances separated by the separating agent layer, wherein regions in which the permeation layer is not laminated are present on a part of the separating agent layer, the separating agent layer exhibits the separability of the target substances and an optical responsiveness to ultraviolet rays, and the permeation layer exhibits an optical responsiveness that is different from those of the target substances and the separating agent layer.
<1> A chromatographic medium having a separating agent layer, which is used to separate target substances, and a permeation layer, which is laminated so as to face the separating agent layer and which is used to enable permeation of the target substances separated by the separating agent layer, wherein a region in which the permeation layer is not laminated is present on a part of the separating agent layer, the separating agent layer exhibits a separating property for the target substances and an optical responsiveness to ultraviolet rays, and the permeation layer exhibits an optical responsiveness that is different from those of the target substances and the separating agent layer.
<2> The chromatographic medium according to <1>, wherein the region in which the permeation layer is not laminated on a part of the separating agent layer is present in a region of the chromatographic medium between a dip end part, which is dipped in a developing solution used to develop the target substances, and half the length of the chromatographic medium in a direction of development.
<3> The chromatographic medium according to <1> or <2>, wherein the permeation layer is laminated in a discontinuous manner in the direction of development of the chromatographic medium.
<4> The chromatographic medium according to any one of <1> to <3>, wherein the permeation layer is laminated in the dotted manner on the separating agent layer.
<5> The chromatographic medium according to <4>, wherein in the permeation layer laminated in the dotted manner, the average diameter of the dots is 0.01 to 5 mm and the pitch between dots is 0.015 to 5 mm.
<6> The chromatographic medium according to any one of <1> to <3>, wherein the permeation layer is laminated on the separating agent layer as band-like rows that intersect a direction of development of the chromatographic medium.
<7> The chromatographic medium according to <6>, wherein bands that form the band-like rows are selected from among straight lines, wavy lines and dashed lines thereof.
<8> The chromatographic medium according to any one of <1> to <7>, wherein the permeation layer is thinner than the separating agent layer.
<9> The chromatographic medium according to any one of <1> to <8>, wherein a separating agent that constitutes the separating agent layer is a separating agent for optical isomers.
<10> The chromatographic medium according to <9>, wherein the separating agent for optical isomers contains a polysaccharide derivative formed of a polysaccharide and one type of group selected from aromatic ester groups, aromatic carbamoyl groups, aromatic ether groups or carbonyl groups that replace some or all of the hydroxyl groups or amino groups in the polysaccharide.
<11> The chromatographic medium according to any one of <1> to <10>, wherein the permeation layer contains a porous material and a fluorescent indicator or coloring reagent as constituent materials.
<12> The chromatographic medium according to <9>, wherein the porous material is silica gel or surface-treated silica gel.
<13> The chromatographic medium according to <11> or <12>, wherein the permeation layer further contains a binder as a constituent material,
<14> The chromatographic medium according to any one of <1> to <13>, wherein scale marks and/or characters are present on the permeation layer.
<15> The chromatographic medium according to <14>, wherein the scale marks and/or characters exhibit an optical responsiveness that are different from those of the permeation layer.
<16> The chromatographic medium according to any one of <1> to <15>, having a base material which faces the separating agent layer or the permeation layer and which supports the chromatographic medium.
<17> The chromatographic medium according to any one of <1> to <16>, wherein the chromatographic medium is plate-shaped, cylindrical or columnar.
<18> A TLC plate having the chromatographic medium according to any one of <1> to <15>, and a base material used to support the chromatographic medium, wherein the chromatographic medium is laminated on a plurality of regions on the base material.
<19> A TLC material formed of the chromatographic medium according to any one of <1> to <15>, and a base material used to support the chromatographic medium.
In the chromatographic medium of the present invention, the permeation layer, which is used to enable the permeation of the separated target substances, is laminated so as to face the separating agent layer, and because this permeation layer exhibits an optical responsiveness that is different from those of the separating agent layer, target substances present in a separating agent layer, which cannot be detected by means of optical responsiveness and which exhibit the same optical responsiveness as the separating agent layer, permeate into the permeation layer and can therefore be detected by means of the optical responsiveness of the target substances that permeate into the permeation layer.
In addition, in the chromatographic medium of the present invention, because a region in which the permeation layer is not laminated is present on a part of the separating agent layer, the target substances appear as spots in this region, and when developing is carried out, it is possible to minimize tailing of the target substances, which can occur as a result of interactions between the separating agent layer and the permeation layer or as a result of the retention of the target substances by the permeation layer.
In addition, because the chromatographic medium of the present invention can achieve the separation and detection of target substances using a single kit, without the need for other components, a complicated procedure is not required to separate and detect the target substances.